The IL-2 receptor and related cytokine/cytokine receptor systems are being studied to understand the T cell immune response in normal and pathologic states. After T-cell activation, the magnitude and duration of the response is controlled in part by the amount of IL-2 produced, levels of IL-2 receptors, and the time course of their induction. IL-2Ra expression is highly expressed by cells infected with HTLV-I, the cause of adult T cell leukemia (ATL). There are 3 chains of the receptor: IL-2Ra, IL-2Rb, and gc, with IL-2Ra and IL-2Rb being regulated at the level of transcription. gc is shared by the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors and is mutated in XSCID. We study the signals induced by these cytokines, particularly STAT proteins and the mechanisms by which they regulate target genes. Given our prior data that Stat5a or Stat5b transgenic mice develop tumors, consistent with STAT5 being implicated in malignant transformation and elevated in a range of human tumors, this has relevance for both normal and pathological states. Moreover, humans and mice with altered STAT protein expression or activation have a range of immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses and other intracelllular pathogens, Th2 differentiation vital in allergic disorders/helminths, and Th17 differentiation vital in inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and that IL-2 induces IL-4 receptor expression in a STAT5-dependent manner and controls priming of cells for Th2 differentiation. Moreover, using genome-wide chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq) analysis, we previously found broad regulation of Th2 differentiation via STAT5A and STAT5B and extended these findings by showing that IL-2 via STAT5 induces IL-12Rb2, which is critical for Th1 differentiation. We also showed that IL-2 via STAT5 also regulates T-bet. Interestingly, IL-2 also inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. We also had reported a critical role of IL-2 in Th9 differentiation, with a direct effect of IL-2 on Th9 differentiation via its induction of STAT5 binding to the Il9 promoter and that IL-2 and IL-21 had opposing actions in Th9 differentiation based on induction of BCL6 by IL-21 but repression by IL-2. In the current year, we have studied the role of new molecules, identified by a computational genomics approach, in Th differentiation. We previously collaborated with Dr. K. Christopher Garcia (Stanford), generating novel IL-2 variants, which represent the first partial agonists for a type 1 cytokine. These next-generation IL-2 variants function as receptor signaling clamps, retaining high affinity for IL-2Rb, inhibiting binding of endogenous IL-2, but their interaction with gc was weakened, attenuating IL-2Rb/gc heterodimerization. We previously showed that one variant, denoted H9-RETR, could prolong survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy with applications to other type 1 cytokines as well. During the past year, we continued our study of these molecules and additionally have studied a new IL-2 partial agonists. IL-21 has broad actions on T- and B-cells, and we previously reported that it also induces apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and that this is inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3, and we previously elucidated roles for STAT1 vs. STAT3 in IL-21 signaling in T cells. Previously, we also demonstrated that IL-21 regulates expression of the Prdm1 gene that encodes BLIMP1 via a response element that depends on STAT3 and IRF4 and subsequently discovered that in contrast to its known ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements (EICEs), IRF4 cooperates with BATF/JUN family proteins to act via novel AP1-IRF composite elements (AICEs) in T cells, as well as in B cells. Studies of AICEs and IRF4/BATF/JUN/STAT3 were continued, and we also had studied the expression of IRF8 as a protective factor for H. Pylori infection with H.C. Morse and contributed to a study by Axel Kallies showing that IL-2 and IL-12 together with BLIMP1 and TBET control effector CD8 T cell differentiation. In other studies, we had studied the biological roles of Egr1 and Egr2 and elucidated some non-immunological roles for Egr1, demonstrating that this transcription factor has a genetic-background dependent effect on eyelid development-- being required for such development on the BALB/c background but not on the C57BL/6 background. In the current year, we extended our studies with H.C. Morse, reporting that the transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses. We also further identified and reported a role for IL-21 in neutrophil biology and elucidated mechanisms related to its cooperative interplay with type I interferon in regulating the innate immune response to S. aureus. Moreover, in tumor model systems, we elucidated mechanisms underlying actions of IL-2 versus IL-21. Previously, we studied the biological significance of STAT5 tetramerization in vivo by generating mice expressing mutant forms of STAT5A and STAT5B that could form dimers but not tetramers and previously reported intricate modeling of the 3-dimensional structure of the tetramer, and we also previously reported the basis for defective NK cell development in the double knockin mice, demonstrating a critical role for STAT5 tetramers for the survival of NK cells. In the current year, we now have extended our studies of tetramers within other cell types as well. We also previously globally characterized super-enhancers regulated by IL-2-activated STAT5 and IL-21-activated STAT3 and their relationship to highly inducible genes and had found that the Il2ra gene contains the most highly ranked STAT5-dependent super enhancer. Using ChIA-PET methodology, we had defined long-distance chromatin interactions and by using CRISPR-Cas9 technology, we functionally dissected the elements of this super-enhancer, providing key new insights into the molecular regulation of the Il2ra in particular and super-enhancers more generally. In the current year, we have significantly extended these studies. Moreover, in collaborative studies with Gary Peltz (Stanford), we also used a haplotype-based computational genomics approach to identify genes involved in Th differentiation. Overall, these studies enhance our understanding of mechanisms by which gc family cytokines regulate gene expression and biological processes and are relevant to normal and pathological immune cell function, including in disease states.